The multistep propagation of discrete intracellular signals allows cells to respond to cues from the extracellular environment. Among the most ubiquitous and well-studied of these are the phosphorylation cascades that culminate in the activation of mitogen-activated protein kinases (MAPKs). The enzymatic activity of MAPKs is markedly influenced by extracellular events. As a rule, p38 MAPK activity is induced by environmental stress (e.g. osmotic shock, hypoxia, heat shock, ultraviolet radiation) and pro-inflammatory stimuli and cytokines such as LPS, IL-1, TGF-beta, and TNF-alpha. The most membrane-proximal enzyme activated in the classic MAPK cascade is a serine/threonine kinase known as a MAPK kinase kinase, or MAPKKK, and the MAPKKKs that lead p38 activation include TAK1, ASK1, and MTK1 (human)/MEKK4 (mouse). Growth Arrest and DNA Damage inducible 45 (Gadd45a) was initially identified as a stress-responsive gene. Our studies of Gadd45&amp;#945;-deficient mice found that they died at an early age of a lupus-like autoimmune disease. Because of the known ability of Gadd45-family proteins to bind and activate MTK1/MEKK4, we asked how the absence of Gadd45a might affect p38 activation. We found that, rather than being hypoactive, p38 was spontaneously active in T-lineage cells. An in-depth analysis of this initial observation led to the following findings by our laboratory: - p38 from antigen receptor-stimulated normal T cells but not B cells robustly autophosphorylates. The autophosphorylation appeared to be on the two canonical activating residues, Thr-180 and Tyr-182. - T cell p38 activation requires Lck and Zap70 but is LAT-independent. - The TCR proximal kinases Lck, Fyn, and Zap70 phosphorylate p38 on Tyr-323, which induces autophosphorylation and enhanced activity toward other substrates. Notably, even p38 lacking Tyr-182 is activated by Tyr-323 phosphorylation. - A Tyr-323 phospho-specific antiserum detects Tyr-323-phosphorylated p38 (p-Tyr-323 p38) in T but not B cells activated via the antigen receptor. - p-Tyr-323 p38 is not detected in Lck+ Zap70- Jurkat T cells, implicating Zap70 as the effector kinase in vivo. - The alternative pathway appeared to be a major mechanism of p38 activation in T cells, because (1) in Jurkat T cells, p38 containing a Y323F substitution was poorly activated in response to anti-TCR compared to wild type (WT), and (2) dual (Thr-180/Tyr-182) phosphorylation of p38 in TCR-stimulated normal resting T cells was almost completely prevented by the p38 inhibitor SB203580, indicating that it is a consequence of autophosphorylation. - p38 from Gadd45&amp;#945;-deficient T cells is spontaneously phosphorylated on Tyr-323. - Gadd45&amp;#945;specifically binds to p38 (whether phosphorylated or not) and inhibits the activity of the p-Tyr-323 form. Importantly, Gadd45a binding does not inhibit the activity of p38 phosphorylated by MKK6 (on Thr-180/Tyr-182). - p38 phosphorylated on Tyr-323 is able to phosphorylate itself in trans; that is, one p38 molecule binds and phosphorylates another. - Auto-trans-phosphorylation involves just Thr-180 and not the canonical Tyr-182 found in the MAPK cascade. - The substrate specificity of mono-phosphosphorylated p38 is different from the di-phosphorylated form. This may explain why this alternative pathway has been evolutionarily conserved in T cells, because the biological effects of these two phosphorylated species would be expected to differ in vivo. - We generated p38 "knock-in" mice in which Tyr-323 is replaced with a Phe (p38YF). Proving the physiologic importance of the alternative pathway, TCR-mediated activation is completely incapable of activating p38 in T cells from these mice. - T cells from p38YF knockin mice are slow to transit from G0 to G1 in the cell cycle upon stimulation via the TCR. Moreover, they make much less interferon-gamma when immunized with Toxoplasma gondii. Therefore, the alternative p38 activation pathway is important for normal T cell proliferation and immune/inflammatory responses. In the past year we have extended our understanding of how p38 is activated in the alternative pathway, and have established an animal model that allows us to explore its importance in normal and pathophysiologic conditions. These recent findings include: - The p38alpha YF knockin mice have been crossed with the GADD45a knockout mice, and we found that the degree of autoimmunity was reduced. This establishes TCR-induced p38 activation as having a key role in autoimmune vasculitis. - We have made p38beta YF knockin mice and have crossed them to the p38alpha YF knockin mice. These animals have more pronounced defects in cell cycle progression and interferon-gamma production. This shows that p38alpha and p38beta are partly redundant, and that both need to be mutated to quantitate the importance of TCR-induced p38 activity in biological responses. - We have found that the GADD45a knockout mice, with elevated p38 activation, are markedly more sensitive to experimental acute encephaolomyelitis, a model of multiple sclerosis, and collagen-induced arthritis, a model of rheumatoid arthritis. We are creating triple mutant mice (GADD45a knockout, p38alpha YF knockin, and p38beta YF knockin) to assess the role of p38 in these diseases. - We have found that T regulatory cells (Tregs) are more active in GADD45a knockout mice and less active in p38alpha YF knocking mice. The mechanism is currently being explored. Our observations establish the alternative p38 pathway as being the major mechanism for the activation of this important kinase in T cells, and that TCR-activated p38 plays an important role in several mouse models of human disease. Our data support the importance of the alternative p38 activation pathway as a molecular target.